CHORUS helps the new direction of green peptide synthesis
Until now, DMF has been considered the gold standard for solid-phase peptide synthesis (SPPS). However, because of its high toxicity and high risk factor, it is classified as a CMR agent (a solvent that is carcinogenic, mutagenic, or toxic to reproduction). On November 22, 2021, the European Union published Regulation (EU) 2021 2030 in its Official Gazette, adding the 76th restriction clause on N,N-dimethylformamide (DMF or DMFA), officially including DMF in the list of restrictions in the REACH regulation. 1 From December 12, 2023, the concentration of the substance itself and the substance containing it is 03% substances or mixtures must not be placed on the market.
Because of this, there is an urgent need to find a suitable alternative to DMF. Peptide-related research groups around the world have conducted extensive research on a variety of alternative and environmentally friendly solvents or mixtures, and have evaluated their potential in SPPS. 2-5
It was found that two alternatives had very promising application prospects, namely N-methyl-2-pyrrolidone (NBP) and a mixture of dimethyl sulfoxide (DMSO) and ethyl acetate (ETOAC). 3-5 Let's take a look at their practical application in SPPS.
When looking for an alternative to DMF, often the polarity and viscosity of the solvent are key factors to consider. 2
DMSO ETOAC mixtures are less dangerous and the polarity of the solvent can be adjusted by varying the ratio of the components to obtain optimal reaction conditions, such as FMOC removal and coupling. However, the use of binary mixtures can also present challenges, such as the use of different solvent ratios for solvent delivery for different reactions in the SPPS process.
Another alternative is NBP, which is a polar passivated green solvent with good performance in SPPs. However, NBP has a high viscosity, making it difficult to accurately deliver amino acids and reagents.
With its patented PurePEP Pathway and induction heating, the PurePEP Chorus Peptide Synthesizer can handle a wide range of solvents precisely and reliably with the freedom to adjust the appropriate temperature. Therefore, we used it to investigate the effect of three different solvent systems (DMF, NBP, and DMSO eTOAC) at two temperatures (room temperature and induction heating to 50) on the crude yield and purity of the three model peptides (Table 1).
Method:
Synthesis
Three different peptides, namely poly-ALA (H-A10K-NH2), acyl carrier protein (65-74, ACP, H-VQAAIDYING-OH) and gonadotropin-releasing hormone (Pyroglu1 gly, G-lhrh, H-ghwsyglrpg-NH2)6,7 were synthesized under normal and high temperature (50) conditions, and different coupling agents were used for diisopropylcarbodiimide Ethyl cyanohydroxyimide acetate (DIC oxyma) and O-(6-chlorobenzo**-1-yl)-N,N,N',n'-Tetramethylurea hexafluorophosphate N-methylmorpholine (HCTU NMM) at 0A 1 mmolar synthesis scale was performed in three different solvents (DMF, NBP, and DMSO Etoac).
With 0Polyala was synthesized on Fmoc-Lys(BOC)-Wang resin, ACP on FMOC-Gly-Wang resin, and G-LHRH on Rink Amide MBHA resin at a ratio of 1 mmol. In the DIC Oxyma conjugation process8, 03 m amino acids (6 equivalents. 4 M oxyma (8 equivalents) and 03 m (DIC, 6 equivalents) for 2 x 15 min. In the HCTU NMM reaction, 03 m amino acids (6 equivalent. 3 m hctu (6 equivalent) and 06 M nmm (12 equivalent) reaction for 2 x 1 min.
FMOC deprotection was performed using 20% PIP for 2 x 2 min at room temperature or induction heated to 50 °C. Regardless of the synthetic solvent used, use TFA:TIS:water (95:2.)5:2.5) Cut the peptide from the resin for 2 h at room temperature, then precipitate in cold ET2O, centrifuge and decant ET2O.
Analytics
Crude peptides were analyzed using the Shimadzu LCMS-2020 with SPD-M20A detector on an ACE Excel 3 C18-PFP, 100 x 30 mm。The mobile phase is water (a) and contains 01% MECN(b) mixture of trans fatty acids. ACP and G-LHRH were dissolved in aqueous acetonitrile (1:1) and analyzed in a gradient of 5 to 70% B over 5 min. Polyala peptides were dissolved in pure water and analyzed over 9 minutes with a gradient of 0-60%B, all at a flow rate of 1 mL min.
Results and discussions
Our research focuses on three peptides that present unique challenges in their synthesis. The first is ACP, an active acyl carrier protein fragment that is recognized as the criterion for judging SPPS due to its tendency to form internal secondary structures during synthesis. 6,7 The second peptide is poly ala, which is known for its self-assembling properties. Finally, we looked at G-LHRH, a peptide that has attracted much attention for its relationship to luteinizing hormone-releasing hormone (LHRH). 7
The selection of these peptides facilitates a comprehensive study of the synthesis process of various peptides in SPPS using different synthesis solvents. After varying the solvent system and temperature, we also tested two different coupling conditions: the carbodiimide-based gold standard coupling system, DIC Oxyma, and the safe and efficient reagent, HCTU NMM. 8,9
Synthesized in DMF
For reference, ACP, G-LHRH, and Poly-Ala were synthesized in DMF using DIC Oxyma or HCTU NMM conjugation reagents, heated to 50°C at room temperature and induction, respectively. The results shown in Table 2 show that DIC Oxyma coupling at 50°C is of higher purity than peptides produced at room temperature, suggesting that the elevated temperature enhances the coupling reaction and reduces the formation of by-products. HCTU NMM conjugation enables extremely fast conjugation (2 x 1 min) and FMOC deprotection (2 x 2 min) steps for rapid synthesis of desired peptides.
Synthesized in NBP
NBP is a bipolar aprotic solvent with high boiling and flash points, and is often used as a solvent for microwave-assisted peptide synthesis. NBP has the advantage of non-production toxicity, resulting in crude peptides of purity comparable to DMF. However, NBP also has the disadvantage of being associated with DMF (0. at 25°C8 cp) compared to NBP (4. at 25°C0 cp), which presents a challenge for accurate transfer of reagents in automated peptide synthesizers.
The PurePEP Chorus Peptide Synthesizer includes a solvent calibration option, which is critical for overcoming the delivery challenges posed by the high viscosity of NBP. With solvent calibration, the user can measure and adjust the amount of liquid delivered to the reaction vessel (RV) as needed.
After calibration, as shown in Table 1, all three peptides proceeded smoothly without any errors when synthesized in a constant temperature chamber in NBP. 4 However, the crude purity of the peptide is slightly lower due to the lower coupling rate in NBP (high viscosity) (27.).4% -52.4%, Table 2).
Synthesized in DMSO Etoac
Recent studies have shown that non-hazardous binary solvent blends such as DMSO Etoac have similar polarity and viscosity profiles to DMF and can be a viable and less hazardous alternative to DMF in SPPS. 2,3 Since polarity plays a crucial role in the results of crude purity in SPPS, the ratio of DMSO etoac was adjusted in this study. Amino acid stock solution (03M) is formulated with low to medium polarity (DMSO Etoac 4:6) and DIC dissolved in Etoac.
On the other hand, the deprotection of FMOC is carried out in a solvent with a relatively high polarity (DMSO Etoac 6:4). Pure eTOAC was used as an end-capping solution, and DMSO Etoac 2:8 was used for post-cycle washing, as detailed in Table 3.
Unlike microwave-assisted peptide synthesis, the Chorus Peptide Synthesizer allows for the flexibility to use a variety of binary solvent systems during SPPS synthesis. As shown in Table 2, the synthesis of all three peptides in DMSO Etoac solution proceeded smoothly at constant temperature, resulting in crude peptide purity ranging from 688% to 854%. When synthesized at 50°C, the average crude purity ranged from 779% to 824%。
Figure 1 shows the UV chromatograms of the crude peptide obtained in DMF and DMSO Etoac, respectively, demonstrating good transferability between the two solvent systems.
Note: Dimethyl sulfoxide (DMSO) is known to cause oxidation of methionine residues during SPPS. However, we successfully synthesized amyloid peptides using a DMSO ETOAC mixture in a nitrogen environment, and the analytical method confirmed that no methionine oxidation occurred (data not shown).
Applicability of the PurePEP Chorus Peptide Synthesizer
Solution calibration
The PurePep Chorus Peptide Synthesizer (Figure 2) is a powerful solid-phase peptide synthesis (SPPS) device that can easily handle highly viscous solvents and solvent mixtures. The system's solvent calibration options ensure precise solvent delivery, regardless of viscosity or composition, which is critical to the success of SPPS. Comparing the SPPS in all three solvents, DMSO Etoac outperformed NBP and DMF for crude purity at constant temperature.
Induction heating
The PurePep Chorus Peptide Synthesizer utilizes precise induction heating and shaking to quickly reach the desired temperature without overheating. This feature enables efficient solid-phase peptide synthesis (SPPS) using a low boiling binary mixture of DMSO Etoac as a solvent at 50 temperatures. Increasing the temperature results in a higher average purity of the three solvents.
The results at a glance
Automated peptide synthesis using NBP and DMSO Etoac instead of dangerous DMF on the PurePep Chorus Peptide Synthesizer yields high-quality peptides.
The process of switching to a green solvent on the PurePep Chorus Peptide Synthesizer can be completed smoothly and quickly.
Since binary solvent mixtures can be inductively heated, DMSO Etoac can be used at high temperatures
Compared to DMF and NMP, DMSO eToac shows higher crude purity at constant temperature and similar purity to DMF at 50°C.
References
1] commission regulation (eu) 2021/2030 of 19 november 2021
2] v. martin et al., green chem 2021, 23, p. 3295
3] s. jadh** et al., green chem 2021, 23, p. 3312
4] j. lopez et al., org process res. dev. 2018, 22, p. 494
5] a. kumar et al., chemsuschem 2020, 13, p. 5288
6] d.m. m. jaradat et.al., green chem 2022, 24, p. 6360
7] v. martin et.al., rsc adv. 2020, 10, p. 42457
8] r. subirós-funosas et.al., eur j org chem 2009, 21, p. 9394
9] c. hood et.al., am biotechnol lab 2008, 26, p. 22